Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

Levay, Konstantin; Cabrera, Jorge L.; Satpaev, Daulet K.; Slepak, Vladlen Z.

doi:10.1073/pnas.96.5.2503

Cited by 92 publications

(99 citation statements)

References 31 publications

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“…Since most of the RGS residues in direct contact with G ␣ are well conserved across the entire family, these residues are unlikely to determine RGS-G protein specificity alone. Rather, additional regulatory proteins, such as effectors (11), G␤ proteins (9,(35)(36)(37)(38), or G proteincoupled receptors (7,39) may be involved. Since five classspecific residues (r77, r117, r121, r122, r124, and r125) cluster above the RGS-G ␣ interface to form an active site common to all members of the family, a reasonable hypothesis is that this is the binding site for additional proteins that mediate specificity (Fig.…”

Section: Discussion Evolutionary Analysis Can Provide Insight Into Rgmentioning

confidence: 99%

A regulator of G protein signaling interaction surface linked to effector specificity

Sowa

Wensel

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the ␣ subunits (G␣) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual G ␣, and GTP hydrolysis rates of different G ␣s can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-G ␣ binding interface and extends to include additional functionally important residues on the surface. One of these is within helix ␣3, two are in ␣5, and three are in the loop connecting ␣5 and ␣6. A cluster of surface residues on G␣ previously identified by ET, and composed predominantly of residues from the switch III region and helix ␣3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the ␥ subunit of Gt␣'s effector, cGMP phosphodiesterase (PDE␥). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-G ␣ complex. Sequence variations in these residues correlate with PDE␥ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.H eterotrimeric G proteins (G ␣␤␥ ) mediate a ubiquitous eukaryotic pathway that converts extracellular signals received by transmembrane serpentine receptors into changes in the concentrations of intracellular ions and small molecule second messengers, thereby controlling vision, cardiac function, and many aspects of neuroendocrine signaling. Upon activation, a receptor catalyzes the exchange of GDP for GTP in the ␣ subunit of a specific G protein (G ␣ ), and either G ␣-GTP or its G ␤␥ partner can interact with a membrane-bound downstream effector protein, leading to amplification of the initial signal. Essential to G protein signaling is the intrinsic temporal regulation of the cascade imposed by G ␣ 's ability to switch back to its inactive form through hydrolysis of GTP. The regulator of G protein signaling (RGS) family of proteins plays a critical role in this process by increasing the intrinsic GTP hydrolysis rate of G ␣ (1-4) and accelerating recovery of the system. Nearly 50 different RGS family members have been identified in eukaryotes thus far, ranging from yeast to humans; and in mammals, individual RGS proteins display distinct expression patterns. However, in general, different types of RGS proteins coexist with a variety of G proteins, leading to the question of how RGS-G ␣ specificity is maintained (5). The crystal structure of the RGS4-G i␣1 complex (6) reveals that the contact residues between the RGS domain and G ␣ are highly conserved in both proteins, implying that in situ RGS-G pr...

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Section: Discussion Evolutionary Analysis Can Provide Insight Into Rgmentioning

confidence: 99%

A regulator of G protein signaling interaction surface linked to effector specificity

Sowa

Wensel

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…However, accurate quantitation of GAP activity is difficult, because rates of GTP hydrolysis are too fast to measure with standard assays (see below). Furthermore, full-length RGS7 was recently found to form a complex with G␤ 5 (29,30), and this complex was shown to have moderate but significant GAP activity toward G␣ o but not G␣ i1 or G␣ i2 (31), despite the fact that it did not seem to favor the physical binding of RGS7 to G␣ o (32). Therefore, it is not clear whether the specificity of full-length RGS7 for G␣ o over G␣ i1 and G␣ i2 is encoded in the RGS domain or it depends on G␤ 5 .…”

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confidence: 99%

Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation

Lan¹,

Zhong²,

Nanamori³

et al. 2000

Journal of Biological Chemistry

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Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits speeding deactivation. G␣ deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of G␣ accompanying GTP hydrolysis and G␣ deactivation on the millisecond time scale. Basal k cat values for G␣ o , G␣ i1 , and G␣ i2 at 20°C were similar (0.025-0.033 s ؊1 ). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of G␣ deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s ؊1 at 3 M, giving a catalytic efficiency of 1.7-2.8 ؋ 10 6 M ؊1 s ؊1 for all three G␣ subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 ؋ 10 6 M ؊1 s ؊1 toward G␣ o , G␣ i2 , and G␣ i1 , respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all G␣ subunits tested, but it is specific for G␣ o over G␣ i1 or G␣ i2 . Furthermore, the specificity of RGS7 for G␣ o does not depend on N-or C-terminal extensions or a G␤ 5 subunit but resides in the RGS domain itself.G-protein 1 -coupled receptor-mediated signal transduction governs many important physiological functions. Upon binding of a ligand, such as light, neurotransmitter, hormone, chemokine, etc., to heptahelical receptors, the heterotrimeric G-proteins composed of ␣, ␤, and ␥ subunits are stimulated to release GDP and bind GTP. In the GTP-bound form, G␣ dissociates from G␤␥ and both interact with downstream effectors. This pathway is terminated when G␣ hydrolyzes the bound GTP, thereby promoting reassociation of G␣ and G␤␥ and returning the system to inactive state (reviewed in Refs. 1-3).Members of the recently described family of proteins, Regulators of G-protein signaling (RGS), act as negative regulators of G-protein function by enhancing GTP hydrolysis by G␣ (4 -6) or by functioning as effector antagonists (7). Although the first RGS protein, Sst2p, was identified in yeast (8, 9), more than 20 variants have been found in mammalian species, all being characterized by a conserved RGS domain of approximately 120 amino acids (reviewed in Refs. 10 -12). The GTPase-activating property (GAP) of RGS proteins has been demonstrated by direct in vitro biochemical studies (13-15). Further studies using GDP-AlF 4 Ϫ suggest that RGS proteins enhance GTP hydrolysis by stabilizing the transition state conformation of G␣ (16), which leads to the crystal structure of the RGS4⅐G␣ i1 complex (17). Mutagenesis analyses have also illustrated the importance of specific residues on the interface between G␣ and RGS proteins (18 -21). Besides being GAPs, many RGS proteins have also been found to serve as links to other cellular signaling pathways through non-RGS domains such as GGL, DEP, DH/PH, and PDZ domains (reviewed in Refs. 11 and 12). The wide expression...

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“…This suggests that the regulatory selectivity observed in cells (18) is achieved by interaction with other signaling molecules. For example, the N terminus of RGS4 confers receptor-selective regulation of G qcoupled responses (19,20); the PDZ domain of RGS12 binds peptides from the C termini of the interleukin-8-RB receptor (21); the GGL domains of RGS6, RGS7, RGS9, and RGS11 selectively bind G␤ 5 (22)(23)(24)(25)(26)(27)(28)(29); and a Dbl-like domain in p115…”

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confidence: 99%

Mechanisms Governing Subcellular Localization and Function of Human RGS2

Heximer

Lim

Bernard

et al. 2001

Journal of Biological Chemistry

118

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RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells. Expression of activated G q increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain is an amphipathic ␣-helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G q . Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear accumulation of RGS2 does not limit its ability to attenuate G q signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes in the nucleus.Many hormones, neurotransmitters, and sensory stimuli elicit specific physiological responses in target tissues by activating receptors that are coupled to heterotrimeric G proteins 1 (1, 2). Activated receptors promote exchange of GTP for GDP on G␣ subunits leading to dissociation of GTP-bound G␣ subunits from G␤␥ heterodimers and activation of downstream effector pathways. Signals are terminated following G␣-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins act as molecular switches to coordinate the wide range of responses elicited by extracellular stimuli.The regulators of G protein signaling (RGS) proteins are a large family that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein ␣ subunits (3-6). The GAP activity of RGS proteins shortens the half-life of active, GTP-bound G␣ subunits and leads to attenuation of a response during prolonged stimulation or accelerated termination of the signal following removal of agonist (7-11). Binding of RGS proteins to active G␣ subunits can also interfere with effector binding, thereby blocking activation and downstream signaling (12). These activities are mediated by the ϳ120-amino acid RGS domain, a conserved feature of this protein family.Genetic studies in budding yeast (13) and Caenorhabditis elegans (14) indicate that eukaryotes express several types of RGS proteins to provide selective regulation of distinct G protein signaling pathways, and a means of differentially regulating a single pathway in response to various physiologic conditions. Similarly, certain mammalian RGS proteins have b...

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Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

Cited by 92 publications

References 31 publications

A regulator of G protein signaling interaction surface linked to effector specificity

A regulator of G protein signaling interaction surface linked to effector specificity

Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation

Mechanisms Governing Subcellular Localization and Function of Human RGS2

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